Biological therapeutics such as proteins are produced in various animal and host cell models. Typically, the product molecule of interest is isolated from the host, host cell, or conditioned medium used to grow the host cell and is purified to remove host proteins and other contaminants before being administered to humans. Sensitive measurement of such host proteins and contaminants is desirable at various points during purification of the product molecule. Currently, techniques for detecting or measuring the host cell proteins and contaminants during purification of product molecules typically involve Enzyme Linked Immunosorbant Assay using antibodies that recognized the host cell protein or contaminant. Such assays typically take at least 5 hours to complete and are typically performed off line, that is samples are removed from the production line and taken to a separate laboratory where the assay is performed. The assays often have a detection limit of about 400 pg/ml.
Significant challenges for a system that detects analytes (e.g., biological agents or biological markers) in liquid media include concentration of the analyte in the media, and transport of the analyte to a sensor surface. For biological applications such as analysis of bioprocess fluids for host cell proteins or contaminants, concentration issues generally arise since the concentrations of such analytes tend to be low. Additionally, biological analytes (e.g., cells, cell fragments and macromolecules such as proteins and nucleic acids) tend to be relatively large; hence, transport issues arise because these larger analytes diffuse in fluid solution very slowly. In addition to cells, cell fragments, and molecules such as proteins and nucleic acids, the detection of small molecule analytes can be a useful marker for diagnosing disease, monitoring drug pharmacokinetics in a patient, screening small molecule libraries for potential drug targets and analyzing bioprocess fluids.
There is a need for improved assays that can quickly detect low concentrations of analyte. In addition, there is a need for improved measurement of analytes including small molecule analytes. Furthermore, a need exists for highly sensitive methods and apparatus for analyzing bioprocess fluids and methods and apparatus that can be used in real time at the production line, or even in line with production in order to streamline the purification process. The ability to analyze bioprocess fluids at line or in line at various stages of the purification process is expected to allow more rapid troubleshooting of production problems, thereby minimizing production downtime.
A key metric for competitive detection is the amount of analyte accumulated on a sensor per unit time. For good performance, the rate of accumulation (and the resulting signal transient) needs to be fast relative to the sensor drift rate. Another key performance metric for an analyte detection system is the degree to which the system can preferentially collect the analyte of interest on the sensor surface. Since many biological samples contain extraneous background components (e.g., other proteins, cells, nucleic acids, dirt), it is necessary to prevent these background components from interfering with the desired measurement. So, a transport method that selectively draws the analyte to the sensor and allows interfering background components to pass by has definite advantages. Such a method used in concert with selective binding of the analyte (e.g., antibody, complimentary DNA strands, etc.) to the sensor surface can deliver high sensitivity measurements for samples with large amounts of extraneous background components relative to the amount of analyte.
Various methods for improving transport of analyte to a sensor surface have been proposed, including filtration, novel flow geometries, acoustic fields, electrical fields (time varying and static) and magnetic fields.
Acoustic excitation has been used to draw cells to field nodes, but it is difficult to use this technique alone to transport material to a surface.
Electrical fields (electrophoresis and dielectrophoresis) have been used to enhance transport but are not universally applicable to all analytes and sample types. They are generally more effective for larger analytes (e.g., cells). Furthermore, the electrical properties of microbes can vary within a given species and strain, making it hard to predict system performance under all intended operating conditions. Sometimes it is necessary to tailor the ionic strength of the sample to improve the performance of the transport. This requirement can conflict with the optimum binding or wash conditions in an assay. Also, electrical fields can dissipate energy and heat conductive fluids (e.g., 0.1 M phosphate buffer solution), which is undesirable since heating can damage the biological analytes.
Immunomagnetic separation (IMS) methods are known in the art for isolating analyte from a sample.